Concanavalin A-Based Sedimentation Assay to Measure Substrate Binding of Glucan Phosphatases.

Glucan phosphatases belong to the larger family of dual specificity phosphatases (DSP) that dephosphorylate glucan substrates, such as glycogen in animals and starch in plants. The crystal structures of glucan phosphatase with model glucan substrates reveal distinct glucan-binding interfaces made of DSP and carbohydrate-binding domains. However, quantitative measurements of glucan-glucan phosphatase interactions with physiologically relevant substrates are fundamental to the biological understanding of the glucan phosphatase family of enzymes and the regulation of energy metabolism. This manuscript reports a Concanavalin A (ConA)-based in vitro sedimentation assay designed to detect the substrate binding affinity of glucan phosphatases against different glucan substrates. As a proof of concept, the dissociation constant (KD) of glucan phosphatase Arabidopsis thaliana Starch Excess4 (SEX4) and amylopectin was determined. The characterization of SEX4 mutants and other members of the glucan phosphatase family of enzymes further demonstrates the utility of this assay to assess the differential binding of protein- carbohydrate interactions. These data demonstrate the suitability of this assay to characterize a wide range of starch and glycogen interacting proteins.

Cooperative Kinetics of the Glucan Phosphatase Starch Excess4.

Glucan phosphatases are members of a functionally diverse family of dual-specificity phosphatase (DSP) enzymes. The plant glucan phosphatase Starch Excess4 (SEX4) binds and dephosphorylates glucans, contributing to processive starch degradation in the chloroplast at night. Little is known about the complex kinetics of SEX4 when acting on its complex physiologically relevant glucan substrate. Therefore, we explored the kinetics of SEX4 against both insoluble starch and soluble amylopectin glucan substrates. SEX4 displays robust activity and a unique sigmoidal kinetic response to amylopectin, characterized by a Hill coefficient of 2.77 ± 0.63, a signature feature of cooperativity. We investigated the basis for this positive kinetic cooperativity and determined that the SEX4 carbohydrate-binding module (CBM) dramatically influences the binding cooperativity and substrate transformation rates. These findings provide insights into a previously unknown but important regulatory role for SEX4 in reversible starch phosphorylation and further advances our understanding of atypical kinetic mechanisms.

Assessing the Biological Activity of the Glucan Phosphatase Laforin.

Glucan phosphatases are a recently discovered family of enzymes that dephosphorylate either starch or glycogen and are essential for proper starch metabolism in plants and glycogen metabolism in humans. Mutations in the gene encoding the only human glucan phosphatase, laforin, result in the fatal, neurodegenerative, epilepsy known as Lafora disease. Here, we describe phosphatase assays to assess both generic laforin phosphatase activity and laforin's unique glycogen phosphatase activity.

Plant α-glucan phosphatases SEX4 and LSF2 display different affinity for amylopectin and amylose.

The plant glucan phosphatases Starch EXcess 4 (SEX4) and Like Sex Four2 (LSF2) apply different starch binding mechanisms. SEX4 contains a carbohydrate binding module, and LSF2 has two surface binding sites (SBSs). We determined KDapp for amylopectin and amylose, and KD for ß-cyclodextrin and validated binding site mutants deploying affinity gel electrophoresis (AGE) and surface plasmon resonance. SEX4 has a higher affinity for amylopectin; LSF2 prefers amylose and ß-cyclodextrin. SEX4 has 50-fold lower KDapp for amylopectin compared to LSF2. Molecular dynamics simulations and AGE data both support long-distance mutual effects of binding at SBSs and the active site in LSF2.

Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates.

Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates.

Structural mechanism of laforin function in glycogen dephosphorylation and lafora disease.

Glycogen is the major mammalian glucose storage cache and is critical for energy homeostasis. Glycogen synthesis in neurons must be tightly controlled due to neuronal sensitivity to perturbations in glycogen metabolism. Lafora disease (LD) is a fatal, congenital, neurodegenerative epilepsy. Mutations in the gene encoding the glycogen phosphatase laforin result in hyperphosphorylated glycogen that forms water-insoluble inclusions called Lafora bodies (LBs). LBs induce neuronal apoptosis and are the causative agent of LD. The mechanism of glycogen dephosphorylation by laforin and dysfunction in LD is unknown. We report the crystal structure of laforin bound to phosphoglucan product, revealing its unique integrated tertiary and quaternary structure. Structure-guided mutagenesis combined with biophysical and biochemical analyses reveal the basis for normal function of laforin in glycogen metabolism. Analyses of LD patient mutations define the mechanism by which subsets of mutations disrupt laforin function. These data provide fundamental insights connecting glycogen metabolism to neurodegenerative disease.

Structures of TraI in solution.

Bacterial conjugation, a DNA transfer mechanism involving transport of one plasmid strand from donor to recipient, is driven by plasmid-encoded proteins. The F TraI protein nicks one F plasmid strand, separates cut and uncut strands, and pilots the cut strand through a secretion pore into the recipient. TraI is a modular protein with identifiable nickase, ssDNA-binding, helicase and protein-protein interaction domains. While domain structures corresponding to roughly 1/3 of TraI have been determined, there has been no comprehensive structural study of the entire TraI molecule, nor an examination of structural changes to TraI upon binding DNA. Here, we combine solution studies using small-angle scattering and circular dichroism spectroscopy with molecular Monte Carlo and molecular dynamics simulations to assess solution behavior of individual and groups of domains. Despite having several long (>100 residues) apparently disordered or highly dynamic regions, TraI folds into a compact molecule. Based on the biophysical characterization, we have generated models of intact TraI. These data and the resulting models have provided clues to the regulation of TraI function.

Phosphoglucan-bound structure of starch phosphatase Starch Excess4 reveals the mechanism for C6 specificity.

Plants use the insoluble polyglucan starch as their primary glucose storage molecule. Reversible phosphorylation, at the C6 and C3 positions of glucose moieties, is the only known natural modification of starch and is the key regulatory mechanism controlling its diurnal breakdown in plant leaves. The glucan phosphatase Starch Excess4 (SEX4) is a position-specific starch phosphatase that is essential for reversible starch phosphorylation; its absence leads to a dramatic accumulation of starch in Arabidopsis, but the basis for its function is unknown. Here we describe the crystal structure of SEX4 bound to maltoheptaose and phosphate to a resolution of 1.65 Å. SEX4 binds maltoheptaose via a continuous binding pocket and active site that spans both the carbohydrate-binding module (CBM) and the dual-specificity phosphatase (DSP) domain. This extended interface is composed of aromatic and hydrophilic residues that form a specific glucan-interacting platform. SEX4 contains a uniquely adapted DSP active site that accommodates a glucan polymer and is responsible for positioning maltoheptaose in a C6-specific orientation. We identified two DSP domain residues that are responsible for SEX4 site-specific activity and, using these insights, we engineered a SEX4 double mutant that completely reversed specificity from the C6 to the C3 position. Our data demonstrate that the two domains act in consort, with the CBM primarily responsible for engaging glucan chains, whereas the DSP integrates them in the catalytic site for position-specific dephosphorylation. These data provide important insights into the structural basis of glucan phosphatase site-specific activity and open new avenues for their biotechnological utilization.

Dimerization of the glucan phosphatase laforin requires the participation of cysteine 329.

Laforin, encoded by a gene that is mutated in Lafora Disease (LD, OMIM 254780), is a modular protein composed of a carbohydrate-binding module and a dual-specificity phosphatase domain. Laforin is the founding member of the glucan-phosphatase family and regulates the levels of phosphate present in glycogen. Multiple reports have described the capability of laforin to form dimers, although the function of these dimers and their relationship with LD remains unclear. Recent evidence suggests that laforin dimerization depends on redox conditions, suggesting that disulfide bonds are involved in laforin dimerization. Using site-directed mutagenesis we constructed laforin mutants in which individual cysteine residues were replaced by serine and then tested the ability of each protein to dimerize using recombinant protein as well as a mammalian cell culture assay. Laforin-Cys329Ser was the only Cys/Ser mutant unable to form dimers in both assays. We also generated a laforin truncation lacking the last three amino acids, laforin-Cys329X, and this truncation also failed to dimerize. Interestingly, laforin-Cys329Ser and laforin-Cys329X were able to bind glucans, and maintained wild type phosphatase activity against both exogenous and biologically relevant substrates. Furthermore, laforin-Cys329Ser was fully capable of participating in the ubiquitination process driven by a laforin-malin complex. These results suggest that dimerization is not required for laforin phosphatase activity, glucan binding, or for the formation of a functional laforin-malin complex. Cumulatively, these results suggest that cysteine 329 is specifically involved in the dimerization process of laforin. Therefore, the C329S mutant constitutes a valuable tool to analyze the physiological implications of laforin's oligomerization.

Solution structure and small angle scattering analysis of TraI (381-569).

TraI, the F plasmid-encoded nickase, is a 1756 amino acid protein essential for conjugative transfer of plasmid DNA from one bacterium to another. Although crystal structures of N- and C-terminal domains of F TraI have been determined, central domains of the protein are structurally unexplored. The central region (between residues 306 and 1520) is known to both bind single-stranded DNA (ssDNA) and unwind DNA through a highly processive helicase activity. Here, we show that the ssDNA binding site is located between residues 381 and 858, and we also present the high-resolution solution structure of the N-terminus of this region (residues 381-569). This fragment folds into a four-strand parallel ß sheet surrounded by α helices, and it resembles the structure of the N-terminus of helicases such as RecD and RecQ despite little sequence similarity. The structure supports the model that F TraI resulted from duplication of a RecD-like domain and subsequent specialization of domains into the more N-terminal ssDNA binding domain and the more C-terminal domain containing helicase motifs. In addition, we provide evidence that the nickase and ssDNA binding domains of TraI are held close together by an 80-residue linker sequence that connects the two domains. These results suggest a possible physical explanation for the apparent negative cooperativity between the nickase and ssDNA binding domain.

Hydroxyurea stimulates the release of ATP from rabbit erythrocytes through an increase in calcium and nitric oxide production.

Hydroxyurea, a proven therapy for sickle cell disease, is known to improve blood flow and reduce vaso-occlusive crises, although its exact mechanism of action is not clear. The objective of this study was to determine if hydroxyurea results in an increase of ATP release from the red blood cell (RBC) via the drug's ability to stimulate nitric oxide (NO) production in these cells. A system enabling the flow of RBCs through microbore tubing was used to investigate ATP release from the RBC. Incubation of rabbit RBCs (7% hct) with 50 microM hydroxyurea resulted in a significant increase in the release of ATP from these cells. This level of ATP release was not detected in the absence of flow. Studies also showed that increments in hydroxyurea and NO (from spermine NONOate) resulted in an initial increase in ATP release, followed by a decrease in this release at higher concentrations of hydroxyurea and the NO donor. Incubation with L-NAME abolished the effect of the hydroxyurea, suggesting that NO production by the RBC was involved. Indeed, in the presence of 50 microM hydroxyurea, the amount of total Ca(2+) measured (by atomic absorption spectroscopy) in a 7% solution of RBCs increased from 363+/-47 ng/ml and 530+/-52 ng/ml. Finally, EPR studies suggest that an increase in nitrosylated Hb in the RBC is only measured for those studies involving hydroxyurea and a Ca(2+)-containing buffer.

Personalized metabolic assessment of erythrocytes using microfluidic delivery to an array of luminescent wells.

The metabolic syndrome is often described as a group of risk factors associated with diabetes. These risk factors include, but are not limited to, such conditions as insulin resistance, obesity, high blood pressure, and oxidant stress. Here, we report on a tool that may provide some clarity on the relationship between some of these associated risk factors, especially oxidant stress and hypertension. Specifically, we describe the ability to simultaneously monitor nicotinamide dinucleotide phosphate (NADPH), reduced glutathione (GSH), and shear-induced adenosine triphosphate (ATP) release from erythrocytes using luminescence detection on a microfabricated device. The measurements are performed by delivering erythrocyte lysate (for the NADPH and GSH measurements, two analytes indicative of oxidative stress) or whole red blood cells (RBCs) (for the determination of ATP release from the cells) to an array of wells that contain the necessary reagents to generate a luminescence emission that is proportional to analyte concentration. A fluorescence macrostereomicroscope enables whole-chip imaging of the resultant emission. The concentrations of each NADPH and GSH contained within a 0.7% erythrocyte solution were determined to be 31.06 +/- 4.12 and 22.55 +/- 2.47 microM, respectively, and the average ATP released from a nonlysed 7% erythrocyte solution was determined to be 0.54 +/- 0.04 microM. Collectively, the device represents a precursor to subsequent merging of microfluidics and microtiter-plate technology for high-throughput assessment of metabolite profiles in the diabetic erythrocyte.

Dynamic monitoring of glutathione in erythrocytes, without a separation step, in the presence of an oxidant insult.

A method for the quantitative determination of the antioxidant form of glutathione (GSH) in red blood cells (RBCs) is described that does not require separation of the analyte of interest from the complex cellular matrix. The measurement portion of the analysis is performed using fluorescence spectrophotometry after monochlorobimane (a recognized probe for GSH) is added to a mixture containing RBCs and glutathione transferase (GST). This method was employed to determine the GSH concentration (0.042 +/- 0.002 mM) in a solution of 1% RBCs obtained from rabbits (n = 6). When spiked with authentic GSH (0.50 micromol), 99.8% of the GSH was recovered. Addition of GST to the sample mixture enabled most measurements to be made after 5-10 min of reaction time. Importantly, a decrease in GSH was measured upon the addition of a recognized oxidant (diamide) to the RBC sample followed by a subsequent return to normal levels of GSH. The ability of the GSH to recover from the oxidant attack occurred in a dose-dependent manner, requiring 30 and 90 min to recover from oxidant insults of 20 and 40 microM diamide, respectively. The antioxidant capabilities of the GSH were able to be monitored in real time, thus providing a method to dynamically monitor the ability of the RBC to maintain homeostasis in a complex matrix.

An altered oxidant defense system in red blood cells affects their ability to release nitric oxide-stimulating ATP.

A novel microflow technique is used to demonstrate that a weakened oxidant defense system found in diabetic erythrocytes leads to decreased levels of deformation-induced release of adenosine triphosphate (ATP) from erythrocytes. Addition of an oxidant to rabbit erythrocytes resulted in a 63% decrease in deformation-induced ATP release before eventually recovering to a value that was statistically equivalent to the initial value. Inhibition of glucose-6-phosphate dehydrogenase prevents recovery from the oxidant attack. Finally, results indicated that the ATP release from the erythrocytes of type II diabetics (91 nM +/- 10 nM) was less than half of that measured from the erythrocytes of healthy controls (190 +/- 10 nM). These data suggest that the antioxidant status of erythrocytes is a critical determinant in the ability of these cells to release ATP, a known nitric oxide stimulus.